Defect pam lock-based crisper / cas12a system, preparation method and application

Abstract

The application provides a CRISPR / Cas12a system based on a defective PAM lock, a preparation method and application, and the application introduces a PAM sequence at the 5' end of an AFB1 aptamer sequence to obtain a functionalized AFB1 aptamer; an error base is introduced on the functionalized AFB1 aptamer to generate a mutant sequence; the functionalized AFB1 aptamer and the mutant sequence are mixed to obtain a complete defective PAM lock recognition probe; the complete defective PAM lock recognition probe is mixed with a sample to be detected and is subjected to first incubation to obtain AFB1-capt; a protein is mixed with a pre-designed crRNA and is subjected to second incubation to obtain a ribonucleoprotein complex; AFB1-capt and the ribonucleoprotein complex are subjected to third incubation to obtain a target system. The application constructs a simple recognition and signal amplification system, reduces the operation difficulty and cost, and reduces background interference.

Description

Technical Field

[0001] This invention relates to the field of electrochemiluminescence sensor technology, and in particular to a CRISPR / Cas12a system based on defective PAM lock, its preparation method, and its application. Background Technology

[0002] Electrochemiluminescence (ECL) is a luminescent phenomenon caused by electrochemical redox reactions. This technology boasts significant advantages such as high sensitivity and low background noise, and its excellent detection performance and rapid response have made it a crucial tool in sensor development. With the advancement of ECL, increasingly more signal amplification strategies are being applied to the construction of ECL sensors, such as rolling circle amplification, CRISPR technology, and enzymatic cycling.

[0003] The CRISPR / Cas system has been applied in electrochemiluminescence biosensors, primarily in the field of nucleic acid detection. Biosensors built based on the CRISPR / Cas system mainly detect target nucleic acids by recognizing crRNA, thereby activating Cas12a to cleave DNA nucleic acids and triggering changes in the ECL signal.

[0004] Some CRISPR / Cas12a systems have been used for the detection of non-nucleic acid targets due to their excellent performance. For example, in existing technologies, the "lock-in" and "unlock-out" states of DNA activators are regulated by aptamers and DNases to achieve quantitative detection of non-nucleic acid targets. Although aptamer sequence design in this existing technology has achieved the detection of non-nucleic acid targets, the complex design steps result in significant drawbacks such as high practical difficulty, cost, and limited universality.

[0005] In existing technologies, non-nucleic acid targets are converted into DNA signals via aptamers, followed by signal amplification through catalytic hairpin assembly (CHA), and fluorescent or colorimetric signal output is achieved using the cleavage activity of CRISPR-Cas12a. However, current methods for constructing electrochemiluminescence sensors using CRISPR for non-nucleic acid targets are complex, and the specificity of CRISPR for nucleic acid sequences limits its direct application to non-nucleic acid targets. Non-nucleic acid targets require signal conversion through biological transduction elements, but the transduction efficiency is low and easily introduces background interference. Unlike nucleic acid targets, which can be rapidly amplified via PCR and RPA, non-nucleic acid targets cannot be directly amplified and require indirect amplification strategies such as aptamer recognition and hybridization chain reaction or chain displacement amplification. However, these steps may introduce non-specific signals, reducing the signal-to-noise ratio. Summary of the Invention

[0006] In view of the above situation, the main objective of this invention is to propose a method for preparing a CRISPR / Cas12a system based on defective PAM locks, so as to solve the above-mentioned technical problems.

[0007] This invention proposes a method for fabricating a CRISPR / Cas12a system based on a defective PAM lock, the method comprising the following steps:

[0008] A PAM sequence was introduced at the 5′ end of the AFB1 aptamer sequence to obtain a functionalized AFB1 aptamer.

[0009] Based on the principle of complementary base pairing, the complete complementary strand of the functionalized AFB1 aptamer is pre-designed, and an incorrect base is introduced at the 3′ end of the complete complementary strand of the functionalized AFB1 aptamer to generate a mutant sequence.

[0010] The functionalized AFB1 aptamer was mixed with the mutant sequence and then heated and slowly cooled to obtain the complete defective PAM lock recognition probe.

[0011] After mixing the intact defect PAM lock identification probe with the sample to be tested, the first incubation was performed to obtain AFB1-capt.

[0012] The LbaCas12a protein was mixed with pre-designed crRNA in buffer and then incubated a second time to obtain the Cas12a / crRNA ribonucleoprotein complex.

[0013] A third incubation was performed on the AFB1-capt and the Cas12a / crRNA ribonucleoprotein complex to obtain the CRISPR / Cas12a system.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] 1. The sensor constructed by the system designed in this invention only needs to replace the aptamer that matches the target to build the corresponding recognition probe. There is no need to make major adjustments to the overall structure of the sensor. It can be flexibly adapted to the detection of various fungal toxins and other non-nucleic acid targets, and has a very broad application prospect.

[0016] 2. This invention constructs a simple identification and signal amplification system of "target-aptamer identification probe-CRISPR", which reduces the difficulty and cost of practical operation and reduces background interference.

[0017] 3. The sensor constructed by the system designed in this invention has a detection limit of 0.2 pg / mL for AFB1 and exhibits a good linear relationship in the concentration range of 1 pg / mL-1 μg / mL. It can accurately capture target pollutants at extremely low concentrations and can still stably perform detection performance in complex real samples (such as Chinese medicinal materials and food), and has potential practical application value. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the synthesis of magnetic ECL nanoprobes;

[0019] Figure 2 Design flowchart for the CRISPR / Cas12a system;

[0020] Figure 3 Flowchart for AFB1 detection using the CRISPR / Cas12a system;

[0021] Figure 4 Transmission electron microscope image of the synthesized nanoparticles;

[0022] Figure 5 The image shows the ECL signal intensity characterization of the nanoparticles.

[0023] Figure 6 The graph shows the change in zeta potential of magnetic nanomaterials after different modifications.

[0024] Figure 7 A schematic diagram of PAM sequence screening;

[0025] Figure 8 This is a graph showing the intensity of electrochemiluminescence.

[0026] Figure 9 This is a schematic diagram of the fluorescence signal intensity after the CRISPR / Cas12a system is activated.

[0027] Figure 10 This is a schematic diagram of the electrochemiluminescence signal intensity;

[0028] Figure 11 Schematic diagram of ECL response at different AFB1 concentrations;

[0029] Figure 12 This is a schematic diagram illustrating the linear relationship between ECL intensity and the logarithm of AFB1 concentration.

[0030] Figure 13 A schematic diagram of the ECL response in the presence of 10 ng / mL AFB1 and other analytes (at a concentration 100 times that of AFB1);

[0031] Figure 14 This is a schematic diagram illustrating the reproducibility of the ECL biosensor. Detailed Implementation

[0032] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0033] Example 1

[0034] like Figure 2 As shown, this embodiment provides a method for fabricating a CRISPR / Cas12a system based on a defective PAM lock, the method comprising the following steps:

[0035] A PAM sequence was introduced at the 5′ end of the AFB1 aptamer sequence to obtain a functionalized AFB1 aptamer.

[0036] Based on the principle of complementary base pairing, the complete complementary strand of the functionalized AFB1 aptamer is pre-designed, and an incorrect base is introduced at the 3′ end of the complete complementary strand of the functionalized AFB1 aptamer to generate a mutant sequence.

[0037] The functionalized AFB1 aptamer (19 μL, 140 nM) was mixed with the mutant sequence (19 μL, 140 nM), and the complete defective PAM lock recognition probe was obtained by heating and then slowly cooling.

[0038] The intact defect PAM lock recognition probe (38 μL, 70 nM) was mixed with the sample to be tested and then incubated for the first time (36.9 °C, 29 min) to obtain AFB1-capt;

[0039] LbaCas12a protein (2.9 μL, 2.5 μg / μL) and pre-designed crRNA were mixed in 1.9 μL of 10×Cas12a-ssDNA buffer II and 12.8 μL of DEPC and then incubated for a second time (36.9 °C, 9 min) to obtain the Cas12a / crRNA ribonucleoprotein complex.

[0040] The AFB1-capt (1.9 μL, 35 nM) and the Cas12a / crRNA ribonucleoprotein complex (17.9 μL) were incubated for the third time (36.9 °C, 9 min) to obtain the CRISPR / Cas12a system.

[0041] Example 2

[0042] This embodiment provides a method for fabricating a CRISPR / Cas12a system based on a defective PAM lock, the method comprising the following steps:

[0043] A PAM sequence was introduced at the 5′ end of the AFB1 aptamer sequence to obtain a functionalized AFB1 aptamer.

[0044] Based on the principle of complementary base pairing, the complete complementary strand of the functionalized AFB1 aptamer is pre-designed, and an incorrect base is introduced at the 3′ end of the complete complementary strand of the functionalized AFB1 aptamer to generate a mutant sequence.

[0045] The functionalized AFB1 aptamer (20 μL, 140 nM) was mixed with the mutant sequence (20 μL, 140 nM), and the complete defective PAM lock recognition probe was obtained by heating and then slowly cooling.

[0046] The intact defect PAM lock recognition probe (40 μL, 70 nM) was mixed with the sample to be tested and then incubated for the first time (37 °C, 30 min) to obtain AFB1-capt;

[0047] LbaCas12a protein (3.0 μL, 2.5 μg / μL) and pre-designed crRNA were mixed in 2.0 μL of 10×Cas12a-ssDNA buffer II and 12.9 μL of DEPC and then incubated for a second time (37 °C, 10 min) to obtain the Cas12a / crRNA ribonucleoprotein complex.

[0048] The AFB1-capt (2.0 μL, 35 nM) and the Cas12a / crRNA ribonucleoprotein complex (18.0 μL) were incubated for the third time (37 °C, 10 min) to obtain the CRISPR / Cas12a system.

[0049] Example 3

[0050] This embodiment provides a method for fabricating a CRISPR / Cas12a system based on a defective PAM lock, the method comprising the following steps:

[0051] A PAM sequence was introduced at the 5′ end of the AFB1 aptamer sequence to obtain a functionalized AFB1 aptamer.

[0052] Based on the principle of complementary base pairing, the complete complementary strand of the functionalized AFB1 aptamer is pre-designed, and an incorrect base is introduced at the 3′ end of the complete complementary strand of the functionalized AFB1 aptamer to generate a mutant sequence.

[0053] The functionalized AFB1 aptamer (21 μL, 140 nM) was mixed with the mutant sequence (21 μL, 140 nM), and the complete defective PAM lock recognition probe was obtained by heating and then slowly cooling.

[0054] The intact defect PAM lock recognition probe (42 μL, 70 nM) was mixed with the sample to be tested and then incubated for the first time (37.1 °C, 31 min) to obtain AFB1-capt;

[0055] LbaCas12a protein (3.1 μL, 2.5 μg / μL) and pre-designed crRNA were mixed in 2.1 μL of 10×Cas12a-ssDNA buffer II and 13.0 μL of DEPC and then incubated for a second time (37.1 °C, 11 min) to obtain the Cas12a / crRNA ribonucleoprotein complex.

[0056] The AFB1-capt (2.1 μL, 35 nM) and the Cas12a / crRNA ribonucleoprotein complex (18.1 μL) were incubated for the third time (37.1 °C, 11 min) to obtain the CRISPR / Cas12a system.

[0057] Example 4

[0058] The present invention also proposes a CRISPR / Cas12a system based on defective PAM locks, wherein the CRISPR / Cas12a system is prepared using the preparation method described in Example 2 above.

[0059] Example 5

[0060] This invention also proposes a detection application of a CRISPR / Cas12a system based on defective PAM locks, using the CRISPR / Cas12a system of Example 2, which is used for the detection of the target analyte AFB1 by electrochemiluminescence.

[0061] The detection steps for the target analyte AFB1 using electrochemiluminescence are as follows:

[0062] like Figure 1 The diagram shown illustrates the synthesis of magnetic ECL nanoprobes. The preparation steps are as follows:

[0063] Will (1g) dissolved in ethylene glycol (20mL), then added (3g) and 1,2-ethylenediamine (10mL) were stirred for 30min until clear. The clear liquid was heated at 200℃ for 6h to obtain ;

[0064] Will (26 mg) was dispersed in ethanol (30 mL) to form the first mixture;

[0065] Add to the first mixture The solution (300 μL, 0.1 M) was used to obtain a second mixture;

[0066] Tetraethyl orthosilicate (800 μL) and ammonia (2 mL, 25%) were added to the second mixture, and a polymerization reaction was carried out to obtain... ;

[0067] Will The solution (6 mL, 6 mg / mL) and ethanol (11.85 mL) were mixed thoroughly in a 100 mL beaker. Then, APTES (150 μL, 0.8%) was slowly added to the beaker. The mixture was stirred at room temperature for 2 hours at a speed of 600 rpm. After the reaction was completed, the desired product was obtained. ;

[0068] Single-stranded ssDNA (ssDNA-COOH) with a 5′ carboxyl-modified end (12.5 μM, 300 μL) was annealed at 90 °C for 10 min. Then, a mixture of NHS (50 μL, 2.3 mg / mL) and EDC (50 μL, 9.76 mg / mL) was added and shaken for 15 min to activate the probe DNA. Then, [further steps were taken]. The solution (300 μL, 4 mg / mL) was thoroughly mixed and reacted at 37 °C for 5 h to obtain... ;

[0069] Verification of the reverse cutting performance of the CRISPR / Cas12a system:

[0070] 18 μL of CRISPR / Cas12a premix (DEPC, Cas12a 13 ng / μL, crRNA 58.3 nM, 10×Cas12a-ssDNA buffer II, FQ-ss DNA Reporter 0.28 μM) was incubated at 37 °C for 10 min to form an unactivated Cas12a / crRNA complex.

[0071] Capt-AFB1 (20 μL, 140 nM) and apt-AFB1 (20 μL, 140 nM) were mixed in Tris-HCl buffer (10 mM Tris-HCl, 50 mM NaCl, 20 mM Tris-HCl, 50 mM NaCl, 20 mM Tris-HCl, 50 mM NaCl, 5 ... Heating to 95℃ for 5 minutes and then naturally cooling to room temperature yields the AFB1 recognition probe.

[0072] Add AFB1 solution (40 μL, 420 nM) to the AFB1 recognition probe and incubate for 30 min to obtain the AFB1 competitive solution;

[0073] Add 2 μL of AFB1 competitive solution (35 nM) to the unactivated Cas12a / crRNA complex, incubate at 37 °C for 10 min, and then measure fluorescence.

[0074] like Figure 3 The diagram shown is a flowchart of the CRISPR / Cas12a system for AFB1 detection. The specific process is as follows:

[0075] Towards Add 18 μL of CRISPR / Cas12a premix to the solution (300 μL), then add 40 μL of AFB1 recognition probe (8 nM) and AFB1 (400 μL, 32 nM), react for 1.5 h, and then perform magnetic separation for 5 min. The supernatant is then discarded.

[0076] Pick Dissolved in PBS (650 μL, 1×), yielding Solution;

[0077] Towards Add 840 μL of PBS (1×, pH 7.4) and 10 μL of tri-n-propylamine to the solution (50 μL) and mix to obtain the detection solution;

[0078] During the detection process, the scanning potential range of the electrochemical workstation was 0.4~1.4V, and the ECL signal generated by the detection solution was recorded. This process was repeated 3 times.

[0079] like Figure 4 The image shown is a transmission electron microscope image of the nanoparticles synthesized in this invention, where A is... TEM image, B is TEM image, C is TEM image, D is TEM images;

[0080] pass Figure 4 It can be observed that the synthesized It is an irregular circle; in contrast, the further synthesized... The regular morphology, uniform size and increased particle size indicate that there is material encapsulation on the particle surface, proving that silicon dioxide successfully encapsulates iron oxide.

[0081] exist Based on this, amino functionalization modification was carried out, from Figure 4 As can be seen, the particles still have a regular morphology and uniform size, and the amino modification on the surface has not destroyed the original core-shell structure.

[0082] pass Figure 4As shown in D in the image, the particles exhibit a denser aggregation and a further increase in particle size, indicating that ssDNA was successfully modified in... surface.

[0083] like Figure 5 As shown, the ECL properties of the synthesized magnetic nanoparticles were also tested in this invention. Figure 5 It can be seen that, Magnetic nanoparticles themselves have almost no electrochemiluminescence activity, so their ECL intensity is almost zero (black line).

[0084] through Packages and After doping, A significant ECL response was observed, confirming Successfully Cover, It has been successfully embedded as a light-emitting probe. middle;

[0085] The generated ECL signal (blue line) is higher than The generated signal is because It contains positively charged amino groups, which make it easy to accumulate near the negatively charged ITO electrode surface;

[0086] The signal is extremely weak because of electrostatic repulsion. It is far removed from the negatively charged ITO electrode surface modified with Nafion.

[0087] The present invention also tested the zeta potential of magnetic nanoparticles with different modifications. , , and Using a, b, c, and d respectively to refer to each other, through Figure 6 The zeta potentials were 8.78, -41.83, -15.75 and -28.4 mV, respectively. These data indicate that the surface modification at different stages was successful and the magnetic ECL probe was successfully synthesized.

[0088] like Figure 7 As shown, this invention screens PAM sequences by performing site-directed mutations at different positions on the complementary strand (capt) sequence and designing seven recognition probes.

[0089] Figure 7 In the diagram, the gray area represents the PAM sequence, the blue area represents the aptamer complementary sequence and the crRNA binding region, and X in the diagram represents the mutation location.

[0090] This invention designs 7 recognition probes, as shown in the table below. The underlined positions in the table are the mutation sites:

[0091] Sequence List:

[0092]

[0093] Based on the base pairing principle (AT / U, GC), this invention designs a complementary sequence for AFB1-apt. Six different defect sites are designed at positions 1-6 of the 3' end of the complementary strand (corresponding to the PAM sequence of AFB1-apt and its adjacent region), i.e., non-base pairing treatment. Each defect site is used to construct a separate ssDNA sequence (denoted as M0-M6). Figure 8 As shown, this invention verified the ability of different recognition probes to activate Cas12a through ECL detection. Considering the ratio of the experimental group to the blank group (signal-to-noise ratio), the M2 recognition probe had the highest signal-to-noise ratio, and the M2 recognition probe was finally selected for subsequent experiments.

[0094] like Figure 9 As shown, the feasibility of this method was verified by fluorescence detection. When AFB1 was absent, the fluorescence intensity was weak; when AFB1 was present, a strong fluorescence signal was observed. This indicates that AFB1 competitively binds to AFB1-apt in the AFB1 recognition probe, causing the release of AFB1-M2 from the probe. This AFB1-M2 then specifically binds to crRNA and activates the trans-cleavage activity of Cas12a, cleaving the ssDNA containing the FAM fluorescent group and the BHQ1 quencher group, resulting in a strong fluorescence signal.

[0095] like Figure 10 As shown, the feasibility of this method was also verified by ECL detection. When AFB1 is absent, A weak ECL signal is generated, while a strong ECL signal is generated when AFB1 is present. This indicates that Cas12a is activated and cleaves the ssDNA on the surface of the nanoparticles, exposing the positively charged amino groups again. This makes the nanoparticles more likely to accumulate near the negatively charged ITO electrode surface, thus generating a strong fluorescence signal.

[0096] Under optimal experimental conditions, this invention measured the electrochemiluminescence intensity of AFB1 at different concentrations and obtained a good linear relationship;

[0097] pass Figure 11 As shown, with the increase of AFB1 concentration, Figure 11In the figures, a and h represent different concentrations of AFB1: a = 0 ng / mL; b = 0.001 ng / mL; c = 0.01 ng / mL; d = 0.1 ng / mL; e = 1 ng / mL; f = 10 ng / mL; g = 100 ng / mL; and h = 1000 ng / mL. These concentrations activate more Cas12a trans-cleavage activity. The activated Cas12a extensively cleaves the negatively charged ssDNA on the surface of the magnetic ECL nanoprobe, exposing more positively charged amino groups on the probe surface. These amino groups are then enriched onto the negatively charged ITO electrode surface through electrostatic attraction. The more probes enriched, the better the surface... The higher the reaction efficiency with the co-reactant TPRA, the stronger the electrochemiluminescence signal produced, ultimately showing a positive correlation between AFB1 concentration and ECL signal. Figure 12 The displayed curve shows the standard operating curve of the ECL sensor constructed using the CRISPR / Cas12a system. It exhibits good linearity in the concentration range of 1 pg / mL to 1 μg / mL, with the regression equation being: Y = 1256lgC + 4892.4 =0.9922), Y is the ECL signal intensity, lgC is the AFB1 concentration, The limit of detection is 0.2 pg / mL (calculated by substituting the mean of the blank sample plus 3 times the standard deviation into the equation).

[0098] To evaluate the specificity of the detection, four different mycotoxins were selected as interfering agents and compared with AFB1, such as... Figure 13 As shown, all interfering devices generated weak ECL signals, while the experimental group AFB1 generated a strong ECL signal. The signal strength of AFB1 mixed with the interfering devices was basically the same as that of AFB1 alone, indicating that the proposed sensor has good selectivity and strong anti-interference ability; Figure 14 As shown, with no target analyte added as a control, the reproducibility of the sensor was studied by repeating the detection of AFB1 (0, 0.001, 0.1 ng / mL) three times. The RSD of each group at the same concentration was calculated to be 0.83%-4.84%, and the deviation within each group was small. These results indicate that the ECL biosensor has high reproducibility.

[0099] To monitor the potential application of the ECL biosensor in actual samples, the content of AFB1 in actual samples was determined using a conventional additive method. The test sample was prepared according to traditional Chinese methods, and the specific steps are as follows:

[0100] Take 3 g of the medicinal material, add 50 mL of ultrapure water and decoct for 30 minutes, then filter.

[0101] Finally, take 3 mL of the obtained filtrate as the test solution;

[0102] Three different concentrations of AFB1 were added to the decoction of dried tangerine peel and lotus seeds and milk, and electrochemiluminescence detection was performed using the developed biosensor.

[0103] The experimental results are shown in Table 1. The recovery rates of dried tangerine peel were 94.00%–106.00%, with relative standard deviations of 3.73%–5.27%; the recovery rates of lotus seeds were 99.00%–108.00%, with relative standard deviations of 1.91%–5.94%; and the recovery rates of milk were 94.67%–105.00%, with relative standard deviations of 1.26%–6.34%. The recovery rates and RSDs of the three different types of actual samples all met the reliability requirements of the detection method, indicating that the developed electroluminescent biosensor can be used for the analysis of aflatoxin in actual samples.

[0104] Table 1. ECL biosensors used for the detection of aflatoxin B1 in real samples.

[0105]

[0106] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0107] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for fabricating a CRISPR / Cas12a system based on a defective PAM lock, characterized in that, The method includes the following steps: A PAM sequence was introduced at the 5′ end of the AFB1 aptamer sequence to obtain a functionalized AFB1 aptamer. Based on the base pairing principle, a fully complementary strand of a functionalized AFB1 aptamer is pre-designed, and an incorrect base is introduced at the 3′ end of the fully complementary strand of the functionalized AFB1 aptamer to generate a mutant sequence. The position of the incorrect base is the first, second, third, fourth, fifth, or sixth base at the 3′ end of the fully complementary strand of the functionalized AFB1 aptamer. The functionalized AFB1 aptamer was mixed with the mutant sequence and then heated and slowly cooled to obtain the complete defective PAM lock recognition probe. After mixing the intact defect PAM lock identification probe with the sample to be tested, the first incubation was performed to obtain AFB1-capt. The LbaCas12a protein was mixed with pre-designed crRNA in buffer and then incubated a second time to obtain the Cas12a / crRNA ribonucleoprotein complex. A third incubation was performed on the AFB1-capt and the Cas12a / crRNA ribonucleoprotein complex to obtain the CRISPR / Cas12a system.

2. The method for fabricating a CRISPR / Cas12a system based on a defective PAM lock according to claim 1, characterized in that, In the process of mixing the functionalized AFB1 aptamer with the mutant sequence and obtaining the complete defective PAM lock recognition probe by heating and then slowly cooling, the amount of functionalized AFB1 aptamer added was 19-21 μL, the concentration of functionalized AFB1 aptamer added was 140 nM, the amount of mutant sequence added was 19-21 μL, and the concentration of mutant sequence added was 140 nM.

3. The method for fabricating a CRISPR / Cas12a system based on a defective PAM lock according to claim 2, characterized in that, After mixing the intact defect PAM lock recognition probe with the sample to be tested and performing the first incubation to obtain AFB1-capt, the amount of intact defect PAM lock recognition probe added was 38-42 μL, and the concentration of intact defect PAM lock recognition probe added was 70 nM.

4. The method for fabricating a CRISPR / Cas12a system based on a defective PAM lock according to claim 3, characterized in that, After mixing the intact defect PAM lock identification probe with the sample to be tested, the first incubation was carried out to obtain AFB1-capt. The temperature of the first incubation was 36.9-37.1℃ and the incubation time was 29-31 min.

5. The method for fabricating a CRISPR / Cas12a system based on a defective PAM lock according to claim 4, characterized in that, After mixing LbaCas12a protein with pre-designed crRNA in buffer, a second incubation was performed to obtain the Cas12a / crRNA ribonucleoprotein complex. The amount of LbaCas12a protein added was 2.9-3.1 μL, the concentration of LbaCas12a protein was 2.5 μg / μL, and the buffer was a mixture of 1.9-2.1 μL of 10×Cas12a-ssDNA buffer II and 12.8-13.0 μL of DEPC.

6. The method for fabricating a CRISPR / Cas12a system based on a defective PAM lock according to claim 5, characterized in that, After mixing LbaCas12a protein with pre-designed crRNA in buffer, a second incubation was performed to obtain the Cas12a / crRNA ribonucleoprotein complex. The temperature of the second incubation was 36.9-37.1℃, and the incubation time was 9-11 min.

7. The method for fabricating a CRISPR / Cas12a system based on a defective PAM lock according to claim 6, characterized in that, In the process of incubating AFB1-capt with the Cas12a / crRNA ribonucleoprotein complex for the third time to obtain the CRISPR / Cas12a system, the amount of AFB1-capt added was 1.9-2.1 μL, the concentration of AFB1-capt was 35 nM, and the amount of Cas12a / crRNA ribonucleoprotein complex added was 17.9-18.1 μL.

8. The method for fabricating a CRISPR / Cas12a system based on a defective PAM lock according to claim 7, characterized in that, In the process of obtaining the CRISPR / Cas12a system by performing a third incubation on the AFB1-capt and Cas12a / crRNA ribonucleoprotein complex, the temperature of the third incubation was 36.9-37.1℃ and the incubation time was 9-11 min.

9. A CRISPR / Cas12a system based on defective PAM locks, characterized in that, The CRISPR / Cas12a system is prepared using the preparation method of any one of claims 1 to 8.

10. A detection application of a CRISPR / Cas12a system based on defective PAM locks, characterized in that, The CRISPR / Cas12a system based on defective PAM lock as described in claim 9 is used for electrochemiluminescence detection of the target analyte AFB1.

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